TY - JOUR
T1 - Neutron-scattering studies reveal further details of the Ca2+/calmodulin-dependent activation mechanism of myosin light chain kinase
AU - Krueger, Joanna K.
AU - Zhi, Gang
AU - Stull, James T.
AU - Trewhella, Jill
N1 - Copyright:
Copyright 2007 Elsevier B.V., All rights reserved.
PY - 1998/10/6
Y1 - 1998/10/6
N2 - Previously, we utilized small-angle X-ray scattering and neutron scattering with contrast variation to obtain the first low-resolution structure of 4Ca2+·calmodulin (CAM) complexed with a functional enzyme, an enzymatically active truncation mutant of skeletal muscle myosin light chain kinase (MLCK). These experiments showed that, upon binding to MLCK, CaM undergoes a conformational collapse identical to that observed when CAM binds to the isolated peptide corresponding to the CaM binding sequence of MLCK. CaM thereby was shown to release the inhibition of the kinase by inducing a significant movement of its CaM binding and autoinhibitory sequences away from the surface of the catalytic core [Krueger, J. K., Olah, G. A., Rokop, S. E., Zhi, G., Stull, J. T., and Trewhella, J. (1997) Biochemistry 36, 6017- 6023]. We report here similar scattering experiments on the CaM·MLCK complex with the addition of substrates; a nonhydrolyzable analogue of adenosine- triphosphate, AMPPNP, and a peptide substrate for MLCK, a phosphorylation sequence from myosin regulatory light chain (pRLC). These substrates are shown to induce an overall compaction of the complex. The separation of the centers-of-mass of the CaM and MLCK components is shortened (by ~12 Å), thus bringing CaM closer to the catalytic site compared to the complex without substrates. In addition, there appears to be a reorientation of CaM with respect to the kinase upon substrate binding that results in interactions between the N-terminal sequence of CaM and the kinase that were not observed in the complex without substrates. Finally, the kinase itself becomes more compact in the CaM·MLCK·pRLC·AMPPNP complex compared to the complex without substrates. This observed compaction of MLCK upon substrate binding is similar to that arising from the closure of the catalytic cleft in cAMP-dependent protein kinase upon binding pseudosubstrate.
AB - Previously, we utilized small-angle X-ray scattering and neutron scattering with contrast variation to obtain the first low-resolution structure of 4Ca2+·calmodulin (CAM) complexed with a functional enzyme, an enzymatically active truncation mutant of skeletal muscle myosin light chain kinase (MLCK). These experiments showed that, upon binding to MLCK, CaM undergoes a conformational collapse identical to that observed when CAM binds to the isolated peptide corresponding to the CaM binding sequence of MLCK. CaM thereby was shown to release the inhibition of the kinase by inducing a significant movement of its CaM binding and autoinhibitory sequences away from the surface of the catalytic core [Krueger, J. K., Olah, G. A., Rokop, S. E., Zhi, G., Stull, J. T., and Trewhella, J. (1997) Biochemistry 36, 6017- 6023]. We report here similar scattering experiments on the CaM·MLCK complex with the addition of substrates; a nonhydrolyzable analogue of adenosine- triphosphate, AMPPNP, and a peptide substrate for MLCK, a phosphorylation sequence from myosin regulatory light chain (pRLC). These substrates are shown to induce an overall compaction of the complex. The separation of the centers-of-mass of the CaM and MLCK components is shortened (by ~12 Å), thus bringing CaM closer to the catalytic site compared to the complex without substrates. In addition, there appears to be a reorientation of CaM with respect to the kinase upon substrate binding that results in interactions between the N-terminal sequence of CaM and the kinase that were not observed in the complex without substrates. Finally, the kinase itself becomes more compact in the CaM·MLCK·pRLC·AMPPNP complex compared to the complex without substrates. This observed compaction of MLCK upon substrate binding is similar to that arising from the closure of the catalytic cleft in cAMP-dependent protein kinase upon binding pseudosubstrate.
UR - http://www.scopus.com/inward/record.url?scp=0032491167&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0032491167&partnerID=8YFLogxK
U2 - 10.1021/bi981311d
DO - 10.1021/bi981311d
M3 - Article
C2 - 9760234
AN - SCOPUS:0032491167
SN - 0006-2960
VL - 37
SP - 13997
EP - 14004
JO - Biochemistry
JF - Biochemistry
IS - 40
ER -